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Immunoassay of digoxin

1) Digoxin immunoassay is used to monitor digoxin levels in patients' serum since digoxin has a narrow therapeutic range and toxic levels can mimic disease symptoms. 2) Various analytical methods are used for digoxin immunoassay including radioimmunoassay, enzyme immunoassay, cloned enzyme donor immunoassay, fluorescence polarization immunoassay, and enzyme multiplied immunoassay. 3) The general procedure involves adding patient serum, labeled digoxin tracer, and antibody to wells before incubating and measuring enzyme activity, with the activity inversely proportional to digoxin concentration in the patient's serum sample.

Education◦
IMMUNOASSAY OF DIGOXIN Presented by – Rashmi M.Pharm 1st sem Pharmacology KUDOPS
Digoxin  Digoxin, also known as digitalis, is a purified cardiac glycoside, extracted from the plant Digitalis lanata.  Digoxin is widely used in the treatment of various heart conditions : • cardiac arrythmias • atrial fibrillation & atrial flutter • cardiac failure that can not be controlled by other medication.
Why we perform digoxin immunoassay?  Digoxin has a narrow range of safe and effective concentrations in serum. Although the therapeutic and toxic concentrations overlap, measurement of digoxin levels helps to maintain effective concentrations and to diagnose and prevent overdosage.
Cont.  Monitoring serum digoxin concentrations, along with careful clinical assessment, is the most effective means of ensuring safe and effective therapy for several reasons:  Studies have shown a relationship between serum digoxin concentrations and clinical signs of toxicity.  Clinical manifestations of digoxin toxicity (cardiac disturbances, gastrointestinal problems , and central nervous system disorders) can mimic those of disease processes.  Concomitant use of other drugs, particularly quinidine, can markedly alter serum digoxin concentrations.
Analytical methods used in immunoassay of digoxin  Methods used to monitor serum digoxin concentrations include : o Radioimmunoassay (RIA), o Enzyme Immunoassay ( EIA) o Cloned Enzyme Donor Immunoassay (CEDIA) o Fluorescence Polarisation Immunoassay (FPIA) o Enzyme Multiplied Immunoassay (EMIT)
Radioimmunoassay (RIA)  The labelled antigen is mixed with the antibody at a concentration that saturates the antigen –binding sites of the antibody.  As the concentration of the unlabeled antigen increases more labelled antigen will be replaced from the binding site.  The sample is washed to remove unbound radioactive drug.  The decrease in the amount of radio labelled antigen bound specific antibody in the presence of the test samples is measured by using a gamma counter to determine the amount of antigen Present in the test sample.  In std Condition, amount of labelled antigen bound to the antibody decreases as the amount of unlabeled antigen increases in sample.
Enzyme Immunoassay ( EIA)  The general principle of this technique is based on the binding of conjugated enzyme molecule with specific antibodies to detect and quantify the presence of either antigens or antibodies in the test sample.  In this method, the label on the tracer is an enzyme.  The catalytic properties of enzymes allows the detection and quantitation of small quantities of the drug.
Cloned Enzyme Donor Immunoassay (CEDIA)  The method uses an antibody to detect the drug to be measured.  A label is used to measure the binding reaction.  It uses genetically engineered fragment of the enzyme β-galactosidase as the label.  This enzyme has two units; an enzyme acceptor and an enzyme donor. These units alone are inactive, but in solution, they become activated and reassemble. As a single unit, they can react with the substrate.  Drug bound to the enzyme donor competes with the drug or metabolite in the sample for the antibody binding site. If the drug bound to the enzyme donor binds to the antibody, it is prevented from reassembling with the enzyme acceptor and activating the enzyme.  If the drug or metabolite is present in the sample, the unbound enzyme donor reassembles with the enzyme acceptor and reacts with the substrate to produce a change in absorbance.
Fluorescence Polarisation Immunoassay (FPIA)  The principle of the assay is that a fluorescent dye (attached to an antigen) can be excited by plane-polarized light at the appropriate wavelength.  The patient sample is incubated with a known quantity of the fluorescent-labeled drug and an antibody specific for the drug. The labeled and unlabeled drugs compete for the binding sites of the antibody. Polarized light is emitted in certain angles depending on whether the fluorescent-labeled drug is bound to antibodies or not. Since this is a competitive assay, the greater the amount of drug in the sample, the lower the amount of fluorescence.  The intensity of polarized light is a measure of concentration of the analyte.
Enzyme Multiplied Immunoassay (EMIT)  Commonly used analytical method for immunoassay of digoxin.  Methodology:  Enzyme multiplied immunoassay is manufactured using recombinant DNA technology, used for the analysis of digoxin and its active metabolites in serum or plasma.  The assay is based on competition between drug in the sample and drug labeled with recombinant glucose-6-phosphatedehydrogenase (rG6PDH) for antibody binding sites. Enzyme activity decreases upon binding to the antibody, so the drug concentration in the sample can be measured in terms of enzyme activity.
General procedure of immunoassay  Specimen Collection and Preparation :  Each assay requires serum or plasma.  Sample volume is instrument-dependent.  Use fresh samples. If samples are to be tested within 8 hours of collection, they may be stored at room temperature (20–25°C).  For transporting, maintain the sample temperature at 2–8°C. Samples can be stored refrigerated at 2–8°C for up to 7 days or stored frozen (- 20°C) for up to 6 months. Repeated freeze-thaw cycles should be avoided.  If the samples contain particulate matter, fibrous material then Vigorously mix sample in a vortex for at least 30 seconds or Centrifuge sample at ≥2000 rpm for 15 minutes.
Cont.  Reagents: • Sample( serum containing digoxin) • Digoxin tracer reagent( digoxin-HRP in a buffer dye) • Rabbit serum antibody(digoxin-ab) • Washing buffer • Signal agent( luminol in buffer).
Procedure:  96-well microplate  Add 0.025ml (25μl) of the appropriate serum specimen into the assigned well.  Add 0.050 ml (50μl) of Digoxin Enzyme Reagent( HRP) to all the wells.  Swirl the microplate gently for 20-30 seconds to mix.  Add 0.050 ml (50μl) Digoxin antibody( rabbit serum) to all wells  Swirl the microplate gently for 20-30 seconds to mix.  Cover and incubate for 30 minutes at room temperature.  Add 0.350ml (350μl) of washing buffer & wash for 3 times.
Cont.  Add 0.100 ml (100μl) of working signal reagent to all wells.  DO NOT SHAKE THE PLATE AFTER THIS ADDITION.  Incubate at room temperature for fifteen (15) minutes.  Read the relative light unit with chemiluminescence microplate reader.  The results should be read within thirty (30) minutes of adding the signal reagent.
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